The poxviridae family members vaccinia and variola (a Class A pathogen) enter mammalian cells, replicate extranuclearly, and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane, and egress away from infected cells towards apposing cells on actin-filled membranous protrusions. We have shown that cell-associated enveloped virions (CEV) utilize Abl- and Src-family tyrosine kinases for actin motility, perhaps permitting motility in a greater range of cell types. Additionally, release of infectious particles (called EEV) from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by Gleevec, an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. EEV are less immunogenic than intracellular forms of the virus, are thought to mediate spread of the virus in and between tissues. Finally, we have demonstrated that Gleevec reduces viral dissemination by five orders of magnitude and promotes survival in lethally infected mice, suggesting a possible utility of this drug in treating smallpox or complications associated with vaccination. The focus of this R01 is on the cellular and viral mechanisms by which Abl- and Src-family kinases mediate actin motility and release of infectious virions, to contribute to viral dissemination. The key questions we address here are: Aim 1: how does the virion recruit and activate tyrosine kinases; Aim 2: what are the cellular and viral targets of the kinases, and how does the phosphorylation of these targets contribute to actin motility and EEV release; and Aim 3: how do actin motility and EEV release contribute to spread of the infection in vivo. Because tyrosine kinases and actin motility mediate movement of a wide range of infectious organisms, we think these questions are of general relevance to virologists and microbiologists. To do this, we will use state of the art microscopy, mass spectroscopy, cell biology, and virology methods together with lead drug compounds in an animal model. These studies will further our understanding of poxvirus dissemination in vitro and in vivo, and may lead to new drug treatments. Moreover, the cell biology paradigm developed here may have applicability to other motile bacterial and viral pathogens. Poxviruses utilize host tyrosine kinases to egress from infected cells. In this application, studies are proposed to explore the mechanism for the novel observation that compounds that inhibit Src- and Abl-family tyrosine kinases (e.g., FDA-approved Gleevec) limit poxvirus egress and release both in vitro and in vivo, a critical prelude for the potential therapeutic use of Gleevec and related compounds as therapeutics for poxviral infections. Project Narrative Poxviruses utilize host tyrosine kinases to egress from infected cells. In this application, studies are proposed to explore the mechanism for the novel observation that compounds that inhibit Src- and Abl-family tyrosine kinases (e.g., FDA-approved Gleevec) limit poxvirus egress and release both in vitro and in vivo, a critical prelude for the potential therapeutic use of Gleevec and related compounds as therapeutics for poxviral infections.